Effect of serum- and leucine-depletion or leucine and/or IGF-1 re-supplementation signaling upstream and downstream of mTOR. (A) Phosphorylation of both upstream activators and downstream effectors of mTORC1 is evident 60 min following treatment with the known mTORC1 stimulators leucine and/or IGF-1. Cells were either serum- and leucine-depleted for 3 h or serum and leucine depleted for 2 h and then re-supplemented with leucine (1mM), IGF-1 (2.67 nM) or the combination of both leucine and IGF-1 for a period of 60 min. Representative blots for PKB phosphorylated on Ser-473 (p-PKB (Ser-473)), PKB total, ERK1/2 phosphorylated on Thr-202/Tyr204 (p-ERK1/2 (Thr-202/Tyr-204)), ERK1/2 total, S6K1, and 4E-BP1 are shown. In all cases the results are representative of at least three separate experiments. (B) Rat 2 fibroblasts were treated as described in Panel A for the indicated period time. Representative blots for PKB phosphorylated on Thr-308 (p-PKB (Thr-308)), PKB phosphorylated on Ser-473 (p-PKB (Ser-473)), PKB total, ERK1/2 phosphorylated on Thr-202/Tyr204 (p-ERK1/2 (Thr-202/Tyr-204)), ERK1/2 total, S6K1, and 4E-BP1 are shown. In all cases the results are representative of at least three experiments.

Repletion of leucine and/or IGF-1 to the culture medium of Rat 2 fibroblasts causes an increased association of eIF2Bε mRNA with actively translating polysome aggregates. Cells were either serum- and leucine-depleted for 3 h (Control) or serum- and leucine-depleted for 2 h and then re-supplemented with leucine (1 mM), IGF-1 (2.67 nM), or the combination of both leucine and IGF-1 for a period of 60 min. (A) Cell supernatants were analyzed by sucrose density gradient centrifugation as described under “Materials and Methods”. In all cases, the results are representative of at least 4 independent experiments. The peaks corresponding to the free (i.e. non-polysome associated) 40S subunits, free 60S subunits, 80S monosomes, and the progressively larger polysome complexes are indicated. Two fractions corresponding to the subpolysomal portion of the gradient (left of the dashed line) and the polysomal portion of the gradient (right of the dashed line) were collected directly into TRIzol for subsequent RNA extraction. (B) RNA from the subpolysomal and polysomal fractions for each treatment group were individually reverse-transcribed to cDNA and analyzed by SYBR Green qRT-PCR using primers specific for the α-, β-, γ-, δ- and ε-subunits of eIF2B as described under "Materials and Methods". Values are expressed as the percent present in the polysomal fraction. The graphical data represents mean values ± SEM (n=4–7 per group). * p<0.05 vs. control. (C) RNA was extracted from cells scraped directly in TRIzol. RNA was reverse-transcribed to cDNA and analyzed by SYBR Green qRT-PCR using primers specific to eIF2Bεor eIF2Bε mRNA. Relative values for eIF2Bε and eIF2Bδ mRNA were normalized to GAPDH mRNA, expressed as a percentage of control and shown as mean values ± SEM (n=9 per group; n.s.).

Pre-treatment of Rat 2 fibroblasts with rapamycin prior to repletion of leucine and IGF-1 represses signaling through mTORC1, the shift of eIF2Bε into actively translating polysomes, and increased incorporation of [³⁵S]Met into eIF2Bε. Cells were treated as described in Fig. 1, Panel A. A subset of cells were pre-treated with the mTORC1 inhibitor rapamycin (100 nM) 30 min prior to re-supplementation with leucine and IGF-1. (A) Representative Western blot analysis of S6K1 and 4E-BP1 phosphorylation status. (B) Representative polysome profile tracings for each treatment group. The peaks corresponding to the free 40S subunits, free 60S subunits, 80S monosomes, and the progressively larger polysome complexes are indicated. Two fractions corresponding to the subpolysomal portion of the gradient (left of the dashed line) and the polysomal portion of the gradient (right of the dashed line) were collected directly into TRIzol for subsequent RNA extraction. (C) RNA was extracted from the subpolysomal and polysomal fractions from each treatment group. The RNA from each fraction was individually reverse-transcribed to cDNA and analyzed by qRT-PCR using primers specific for the ε-subunit of eIF2B. Values are expressed as the percent present in the polysomal fraction. The graphical data represents mean values ± SEM (n=6 per group). * p<0.001 vs. control; † p<0.01 vs. L+I. (D) Incorporation of [³⁵S]Met/Cys into eIF2Bε was measured in eIF2Bε immunoprecipitates as described under “Materials and Methods”. An autoradiograph from a typical experiment is shown as an inset. Within each experiment, three separate dishes of cells was analyzed per condition, and three such experiments were performed. * p<0.05 vs. control; † p<0.05 vs. L+I‥

Overexpression of FLAG-Rheb activates mTORC1 signaling and is sufficient to increase eIF2Bε̣mRNA polysome association. Rat 2 fibroblasts were transfected with Lipofectamine 2000 for 5 h and then allowed to recover overnight. Cells were then serum-starved for 3 h prior to analysis. (A) Western blot analysis of exogenously expressed (Flag) and endogenous Rheb, S6K1 and 4E-BP1. Blots representative of 3 independent experiments are shown. (B) RNA was extracted from both the subpolysomal and polysomal fractions, reversed transcribed, and analyzed for relative eIF2Bε and GAPDH mRNA abundance respectively using qRT-PCR. Values are expressed as the percent present in the polysomal fraction. The graphical data represents mean values ± SEM (n=6 per group). * p<0.001 vs. control. (C) Total RNA was reverse-transcribed and analyzed by SYBR Green qRT-PCR using primers specific for eIF2Bε. Relative values for eIF2Bε were normalized to GAPDH, expressed as a percentage of control and shown as mean values ± SEM (n=6 per group; n.s). (D) Incorporation of [³⁵S]Met/Cys into eIF2Bε was measured in eIF2Bε immunoprecipitates as described under “Materials and Methods”. An autoradiograph from a typical experiment is shown as an inset. Within each experiment, three separate dishes of cells was analyzed per condition, and three such experiments were performed. * p<0.05 vs. control. (E) Rat 2 fibroblasts transfected with either the pRK7 or FLAG-Rheb plasmid were serum starved as described in Panel A and rapamycin was added to one-half of the culture dishes as described in the legend to Fig. 3. Rheb expression and S6K1 phosphorylation were assessed by Western blot analysis. Blots representative of 3 independent experiments are shown. (F) RNA from the each condition depicted in Panel E was subjected to sucrose density gradient centrifugation followed by isolation of RNA from the subpolysomal and polysomal fractions. Total RNA was analyzed by qRT-PCR as described in Panel C. Within each experiment, three separate dishes of cells was analyzed per condition, and three such experiments were performed. Relative values for eIF2Bε were normalized to GAPDH, expressed as a percentage of control and shown as mean values ± SEM. * p<0.01 vs. control

Decreased eIF2Bε expression leads to reduced cellular proliferation rate, eIF2B activity, and global rates of protein synthesis. Rat 2 cells at 50% confluency were transfected with siRNA targeted against eIF2Bε or a scrambled (control) siRNA as described under “Materials and Methods”. Twenty-four hours later, 5×10⁴ cells were equally plated into 100mm dishes. (A) Protein lysates were prepared from cells at 0 and 72h after plating and 50 µg of protein was subjected to Western blot analysis using anti-eIF2Bε antibodies. (B) eIF2B guanine nucleotide exchange activity was measured as described under “Materials and Methods" and is expressed as a percentage of the activity observed in homogenates of cells transfected with the control siRNA. Within each experiment, three separate dishes of cells were analyzed per condition, and three such experiments were performed. (C) Global rates of protein synthesis were measured as described under “Materials and Methods” and are expressed as a percentage of the rate observed in cells transfected with the control siRNA. Within each experiment, three separate dishes of cells were analyzed per condition, and seven such experiments were performed. * p<0.05 vs control; † p<0.01 vs control. (D) For the following 72 hrs, cells were counted at 24 hr intervals and are compiled from 3 separate experiments with 0 hr being the time of plating.